Biomedical subjects

Marcus R Clark

Publications and source records attributed to Marcus R Clark.

15 recordsLinked to original sources

PubMed2026

Spatial proximity sequencing maps developmental dynamics in the germinal center.

Spatial profiling of proteins and protein interactions facilitates understanding of cell functions within tissues and is essential for studies in signaling, immunity, and cancer. We present spatial proximity sequencing (Sprox-seq) for simultaneous profiling of surface proteins, protein complexes, and mRNAs, recording the tissue location of each molecule. Sprox-seq profiled 32 proteins, 528 pairwise interactions, and thousands of mRNAs with spatial resolution across human tonsils and germinal centers. Mapping tissue-wide protein interactions recapitulated RNA-defined tissue architecture but also revealed higher interaction complexity in the light zone. Protein-interaction trajectories uncovered a B cell state transition distinct from that inferred by RNA. Integrated protein-complex and mRNA analysis related spatially enriched complexes with mitotic pathways. Sprox-seq captured cell-cell interactions, such as B cell-follicular dendritic cell interactions mediated by the receptor complex VLA-4-VCAM1. Sprox-seq provides a spatially resolved multi-modal view of cell states and an integrated study of protein and cellular interactions across tissues.

Humans↗

PubMed2006

A unique function for cyclin D3 in early B cell development.

During hematopoiesis, stem cell proliferation is dependent on expression of the D-type cyclins. However, little is known about how each cyclin D contributes to the development of specific hematopoietic lineages. Here, analysis of Ccnd1(-/-), Ccnd2(-/-), Ccnd3(-/-) and Ccnd2(-/-)Ccnd3(-/-) mice showed that cyclin D3 was uniquely required for the development of pre-B cells. Transcription of Ccnd3 was dependent on expression of the common gamma-chain. In contrast, expression of the pre-B cell receptor and activation of 'downstream' signaling pathways prevented proteasome-mediated degradation of cyclin D3. Cyclin D3 has a key function in B cell development by integrating cytokine and pre-B cell receptor-dependent signals to expand the pool of pre-B cells that have successfully rearranged immunoglobulin heavy chain.

Animals↗

PubMed2006

HS1 functions as an essential actin-regulatory adaptor protein at the immune synapse.

HS1, the leukocyte-specific homolog of cortactin, regulates F-actin in vitro and is phosphorylated in response to TCR ligation, but its role in lymphocyte activation has not been addressed. We demonstrate that HS1-deficient T cells fail to accumulate F-actin at the immune synapse (IS) and, upon TCR ligation, form actin-rich structures that are disordered and unstable. Early TCR activation events are intact in these cells, but Ca2+ influx and IL-2 gene transcription are defective. Importantly, HS1 tyrosine phosphorylation is required for its targeting to the IS and for its function in regulating actin dynamics and IL-2 promoter activity. Phosphorylation also links HS1 to multiple signaling proteins, including Lck, PLCgamma1, and Vav1, and is essential for the stable recruitment of Vav1 to the IS. Taken together, our studies show that HS1 is indispensable for signaling events leading to actin assembly and IL-2 production during T cell activation.

Actins↗

PubMed2006

B cell antigen receptor signaling and internalization are mutually exclusive events.

Engagement of the B cell antigen receptor initiates two concurrent processes, signaling and receptor internalization. While both are required for normal humoral immune responses, the relationship between these two processes is unknown. Herein, we demonstrate that following receptor ligation, a small subpopulation of B cell antigen receptors are inductively phosphorylated and selectively retained at the cell surface where they can serve as scaffolds for the assembly of signaling molecules. In contrast, the larger population of non-phosphorylated receptors is rapidly endocytosed. Each receptor can undergo only one of two mutually exclusive fates because the tyrosine-based motifs that mediate signaling when phosphorylated mediate internalization when not phosphorylated. Mathematical modeling indicates that the observed competition between receptor phosphorylation and internalization enhances signaling responses to low avidity ligands.

Amino Acid Motifs↗

PubMed2005

Spi-1 and Spi-B control the expression of the Grap2 gene in B cells.

The Ets family members Spi-1 and Spi-B have been implicated in the regulation of genes important for B cell antigen receptor (BCR) signaling. Mice deficient in Spi-B exhibit reduced B cell proliferation in response to BCR cross-linking and impaired T cell-dependent immune responses. This defect is exacerbated in the presence of Spi-1 haplo-insufficiency (Spi1+/- SpiB-/-). Tyrosine phosphorylation and calcium mobilization induced by BCR engagement is diminished in Spi1+/- SpiB-/- B lymphocytes, although many key BCR signaling proteins are expressed, suggesting that Spi-1 and Spi-B regulate expression of additional, unidentified signaling molecules. We now demonstrate that expression of the adaptor protein Grap2 is impaired in Spi1+/- SpiB+/- and Spi1+/- SpiB-/- B lymphocytes. Analysis of two alternate murine Grap2 promoters revealed a functionally important Spi-1 and Spi-B DNA binding element located in the downstream promoter. Ectopic expression of Grap2 in Grap2-deficient B cells reduced the recruitment of BLNK to Igalpha and the phosphorylation of specific substrates. Regulation of BLNK recruitment was dependent upon the Grap2 proline-rich domain, while modulation of phosphorylation was dependent upon both the proline-rich and SH2 domains. These data indicate that Spi-1 and Spi-B directly regulate the expression of Grap2 and that Grap2 functions to modulate BCR signaling, but that reduced Grap2 expression is unlikely to account for the BCR signaling defects observed in Spi1+/- SpiB-/- B cells.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Membrane-targeted peptides derived from Igalpha attenuate B-cell antigen receptor function.

Within the B-cell antigen receptor (BCR), heterodimers of Igalpha/Igbeta couple the receptor to intracellular signaling pathways. In the resting state, Igalpha associates with Src-family tyrosine kinases (SFTKs) which contain some basal activity. Upon engagement of the receptor, the SFTKs phosphorylate tyrosine residues in the BCR that recruit and activate the tyrosine kinase Syk, initiating signaling pathways. To test the hypothesis that disrupting the association between the resting receptor and the SFTKs would attenuate both basal and induced receptor activities, we expressed non-phosphorylatable membrane-targeted analogs of Igalpha (Igalpha/M) or Igbeta (Igbeta/M) in B lymphocytes. Both Igalpha/M and Igbeta/M inhibited BCR-induced calcium mobilization, but only Igalpha/M was able to diminish tyrosine phosphorylation. In an immature B-cell line, Igalpha/M attenuated both receptor-induced and basal apoptosis. Taken together, these data demonstrate the importance of the resting receptor complex and suggest therapeutic strategies for regulating receptor-mediated functions.

Antigens, CD↗

PubMed2004

Selection of B lymphocytes in the periphery is determined by the functional capacity of the B cell antigen receptor.

Within the B cell antigen receptor (BCR), the cytoplasmic tails of both Igalpha and Igbeta are required for normal B cell development and maturation. To dissect the mechanisms by which each tail contributes to development in vivo, Igbeta(-/-) mice were reconstituted with retroviruses encoding either wild-type Igbeta, an Igbeta molecule lacking a cytoplasmic tail (Igbeta(deltaC)) or one in which the cytoplasmic tail was derived from Igalpha (Igbeta(Calpha)). All constructs rescued B cell development and generated immature B cell populations in the bone marrow with similar expression levels of both Igbeta and membrane-bound IgM. In the periphery, receptor-surface density was inversely proportional to the number of Igalpha tails in the BCR. Although peripheral-surface-receptor levels differed, splenic B cells expressing either Igbeta or Igbeta(Calpha) responded similarly to stimulation through the BCR. Analysis of membrane-bound IgM and Igbeta expression revealed that peripheral-receptor expression was primarily determined by positive selection between the bone marrow and peripheral immature B cell populations. These data indicate that B cells are selected into the periphery on the basis of a common level of antigen responsiveness.

Animals↗

PubMed2004

B-cell antigen receptor signaling requirements for targeting antigen to the MHC class II presentation pathway.

The ability of B lymphocytes to capture, process and present antigens to T cells is requisite for normal humoral immune responses and contributes to the pathogenesis of both B- and T-cell-mediated autoimmune diseases. B lymphocytes preferentially capture polyvalent antigens, which are capable of eliciting a coordinated series of cellular responses that ensure that even low-affinity antigens are productively captured. Polyvalency not only accelerates transit through the endocytic pathway but also induces a reorganization of the antigen-processing compartment, activates degradative pathways and determines how antigenic peptides are presented to T cells. Similar changes are observed in maturing dendritic cells, indicating that some cellular responses to foreign antigens are conserved.

Animals↗

PubMed2003

B-cell antigen-receptor signalling in lymphocyte development.

Signalling through the B-cell antigen receptor (BCR) is required throughout B-cell development and peripheral maturation. Targeted disruption of BCR components or downstream effectors indicates that specific signalling mechanisms are preferentially required for central B-cell development, peripheral maturation and repertoire selection. Additionally, the avidity and the context in which antigen is encountered determine both cell fate and differentiation in the periphery. Although the signalling and receptor components required at each stage have been largely elucidated, the molecular mechanisms through which specific signalling are evoked at each stage are still obscure. In particular, it is not known how the pre-BCR initiates the signals required for normal development or how immature B cells regulate the signalling pathways that determine cell fate. In this review, we will summarize the recent studies that have defined the molecules required for B-cell development and maturation as well as the theories on how signals may be regulated at each stage.

Animals↗

PubMed2003

Molecular mechanisms of B cell antigen receptor trafficking.

B lymphocytes are among the most efficient cells of the immune system in capturing, processing, and presenting MHC class II restricted peptides to T cells. Antigen capture is essentially restricted by the specificity of the clonotypic antigen receptor expressed on each B lymphocyte. However, receptor recognition is only one factor determining whether an antigen is processed and presented. The context of antigen encounter is crucial. In particular, polyvalent arrays of repetitive epitopes, indicative of infection, accelerate the delivery of antigen to specialized processing compartments, and up-regulate the surface expression of MHC class II and co-stimulatory molecules such as B7. Recent studies have demonstrated that receptor-mediated signaling and receptor-facilitated peptide presentation to T cells are intimately related. For example, rapid sorting of endocytosed receptor complexes through early endosomes requires the activation of the tyrosine Syk. This proximal kinase initiates all BCR-dependent signaling pathways. Subsequent entry into the antigen-processing compartment requires the tyrosine phosphorylation of the BCR constituent Igalpha and direct recruitment of the linker protein BLNK. Signals from the BCR also regulate the biophysical and biochemical properties of the targeted antigen-processing compartments. These observations indicate that the activation and recruitment of signaling molecules by the BCR orchestrate a complex series of cellular responses that favor the presentation of even rare or low-affinity antigens if encountered in contexts indicative of infection. The requirement for BCR signaling provides possible mechanisms by which cognate B:T cell interactions can be controlled by the milieu in which antigen engagement occurs.

Peptides↗

PubMed2002

Receptor-facilitated antigen presentation requires the recruitment of B cell linker protein to Igalpha.

Ags that cross-link the B cell Ag receptor are preferentially and rapidly delivered to the MHC class II-enriched compartment for processing into peptides and subsequent loading onto MHC class II. Proper sorting of Ag/receptor complexes requires the recruitment of Syk to the phosphorylated immunoreceptor tyrosine-based activation motif tyrosines of the B cell Ag receptor constituent Igalpha. We postulated that the Igalpha nonimmunoreceptor tyrosine-based activation motif tyrosines, Y(176) and Y(204), contributed to receptor trafficking. Igalpha(YDeltaF(176,204))/Igbeta receptors were targeted to late endosomes, but were excluded from the vesicle lumen and could not facilitate the presentation of Ag to T cells. Subsequent analysis demonstrated that phosphorylation of Y(176)/Y(204) recruited the B cell linker protein, Vav, and Grb2. Reconstitution of Igalpha(YDeltaF(176,204))/Igbeta with the B cell linker protein rescued both receptor-facilitated Ag presentation and entry into the MHC class II-enriched compartment. Thus, aggregation accelerates receptor trafficking by recruiting two separate signaling modules required for transit through sequential checkpoints.

Adaptor Proteins, Signal Transducing↗

PubMed2002

A point mutation in the constant region of Ig lambda1 prevents normal B cell development due to defective BCR signaling.

Surface expression of B cell antigen receptors (BCRs) containing Ig and Igalpha/Igbeta generates signals required to transit discrete developmental checkpoints. The mechanism by which BCR components collaborate to initiate signals is still unclear. The expression of Iglambda1 in SJL mice is 50-fold lower than in other strains. Here, we demonstrate by gene targeting that a point mutation, which changes a glycine to a valine in the lambda1 constant region, is responsible for this defect. In vitro experiments show that Ig receptors bearing this mutation, while expressed normally, are deficient in signaling. These findings reveal a direct involvement of the Ig light chain (IgL) in B cell signaling and development beyond the requirement of light chains for BCR assembly.

Animals↗

PubMed2002

The direct recruitment of BLNK to immunoglobulin alpha couples the B-cell antigen receptor to distal signaling pathways.

Following B-cell antigen receptor (BCR) ligation, the cytoplasmic domains of immunoglobulin alpha (Ig alpha) and Ig beta recruit Syk to initiate signaling cascades. The coupling of Syk to several distal substrates requires linker protein BLNK. However, the mechanism by which BLNK is recruited to the BCR is unknown. Using chimeric receptors with wild-type and mutant Ig alpha cytoplasmic tails we show that the non-immunoreceptor tyrosine-based activation motif (ITAM) tyrosines, Y176 and Y204, are required to activate BLNK-dependent pathways. Subsequent analysis demonstrated that BLNK bound directly to phospho-Y204 and that fusing BLNK to mutated Ig alpha reconstituted downstream signaling events. Moreover, ligation of the endogenous BCR induced Y204 phosphorylation and BLNK recruitment. These data demonstrate that the non-ITAM tyrosines of Ig alpha couple Syk activation to BLNK-dependent pathways.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Modified anti-CD3 therapy in psoriatic arthritis: a phase I/II clinical trial.

OBJECTIVE: Treatment of autoimmune diseases with therapies that tolerize pathogenic lymphocytes may obviate the need for longterm global immunosuppression. In vitro, non-Fc receptor binding derivatives of anti-murine CD3 monoclonal antibodies tolerize type 1 T cells and stimulate type 2 T cells. Recently, a humanized non-FcR binding derivative of the anti-human CD3 Mab OKT3, huOKT3gamma1(ala-ala), has been described. We hypothesized that this Mab may be safe and efficacious in the treatment of type 1 T lymphocyte mediated chronic autoimmune diseases such as psoriatic arthritis (PsA). METHODS: In a Phase I/II trial, 7 patients with PsA were treated with escalating daily doses of huOKT3gamma1(ala-ala) for 12 to 14 days. Number of tender and swollen joints and a visual analog pain scale were used to rate disease activity at entry and Day 30 and Day 90 after treatment. RESULTS: At Day 30, 6 of 7 patients had > or = 75% improvement in the number of inflamed joints and an average 63% improvement on the patient pain scale. Two of 6 responders had sustained improvement at Day 90. No patient treated with an initial dose < or = 1 mg had significant side effects, nor did they have detectable increases in serum cytokines. One patient treated with 4 mg without escalation developed mild cytokine release symptoms associated with elevation of interleukin 10. Transient T cell depletion occurred following treatment with the maximum dose of 4 mg, which resolved by Day 30. Antiidiotypic antibodies developed in 2 patients; however, there was no concurrent decrease in efficacy. CONCLUSION: These data indicate that huOKT3gamma1(ala-ala) may be useful in treating PsA.

Adolescent↗

PubMed2002

Cooperative interaction of Ig(alpha) and Ig(beta) of the BCR regulates the kinetics and specificity of antigen targeting.

Following the binding of antigens, the BCR transduces signals and internalizes antigens for processing and presentation, both of which are required for initiating an effective antibody response. The BCR, consisting of membrane Ig and Ig(alpha)/Ig(beta) heterodimer, facilitates antigen processing by accelerating antigen targeting to the processing compartment. Previous reports showed that Ig(alpha) or Ig(beta) alone is competent for internalizing antigens. However, both Ig(alpha) and Ig(beta) are required for BCR-enhanced antigen presentation. Using chimeric proteins containing the extracellular and transmembrane domains of human platelet-derived growth factor receptor fused with the cytoplasmic domain of Ig(alpha) or Ig(beta), we studied the roles of the cytoplasmic tails of Ig(alpha) and Ig(beta) in BCR-mediated antigen transport. The Ig(beta) chimera rapidly moves through the endocytic pathway to lysosomes, while the Ig(alpha) chimera slows down this movement. The Ig(alpha), but not the Ig(beta) chimera, is required for an increase in the turnover rate of the chimeras in response to stimulation. Only when Ig(alpha) and Ig(beta) chimeras are co-expressed do the chimeras rapidly and specifically target antigens to the processing compartment. These findings suggest that Ig(alpha) and Ig(beta) play distinct roles in BCR trafficking, and the cooperative interaction of Ig(alpha) and Ig(beta) controls and regulates the kinetics and specificity of antigen targeting.

Animals↗